L1 - Cell Cycle Control

Function of Cell Division

Embryonic Development
Tissue regeneration - to replace naturally dying cells
Homeostasis - to repair

The cell cycle

The basis of replication, development and growth
Highly conserved across eukaryotes
Requires intricate coordination
Interphase + M phase
Occurs every 24h (ish)
Not every cell goes through it

When the cell grows it needs to make more:

Proteins
RNA
DNA
Lipids
Organelles

Interphase

The growth phase
G1 (Gap 1) + S (Synthesis) + G2 (Gap 2)
Over 90% of time spent in this phase
The chromosomes are relaxed and long

M phase

The division phase
Mitosis + Cytokinesis

Division of cellular components

Most cellular content is divided roughly in two, except for DNA which divided exactly in two

Quiescence

G0
Reversible cell cycle exit
Temporarily leaving the cell cycle

Senescence

Permanent cell cycle exit
In specialised or damaged cells
To prevent cancer by stopping growing entirely

G1 Phase

Gap 1
Growth phase
Synthesis of RNA and proteins
Where the cycle starts

S phase

Synthesis
When DNA replication occurs
Cannot finish until all DNA is replicated

G2 phase

Skipped by some cells
DNA repair
Cell prepares for mitosis
At the end of this stage the cell has 2 copies of each 46 chromosomes

Mitotic cells in culture

Round up
In order to go through division cells detach and look round rather than flat

Mitosis

DNA is partitioned equally during mitosis with the help of centromeres
Results in two diploid daughter cells, identical to the parent
Prophase
Prometaphase
Metaphase
Anaphase
Telophase

Prophase

Chromosomes condense
Spindle aparatus begins to form
The two sister chromatids lie togehter, attached at the centromere

Prometaphase

Nuclear envelope breaks down
Microtubules contact chromosomes at kinetochores

Metaphase

Chromosomes complete migration to the middle of the cell (equatorial plane)
Most highly condensed chromosomes

Anaphase

Sister chromatids separate into daughter chromosomes and are pulled to opposite poles of the spindle apparatus, centromere first
End with 92 seperate chromosomes, half near each

Telophase

The nuclear envelope re-forms and chromosomes condense
Spindle fibres disappear

Centrosome

The principal microtubule organising centre (MTOC)
Duplicated and divided exactly once per cell cycle
After duplication the centrosomes migrate to either ends of the cell
Most cells only have one centrosome, but some (e.g. cilia) have multiple

The Centrosome Cycle

Centrosome cycle runs at the same time as cell cycle - shares many features

Cell cycle regulation

Cells cannot return to the previous state after a restriction point
Checkpoints maintain directionality and as quality control
Mostly involving PTMs, small and reversible, covalently attached, needing an enzyme and being veru strong

Kinase

Phosphorylate the target

Phosphatase

Dephosphorylate the target

Cyclin

Non enzymatic protein
Levels of cyclin increase and decrease throughout the cell cycle
- Through protein synthesis and cleavage

Cdk

Cyclin dependent kinase
A kinase that can phosphorylate targets
Requires cyclin to function (only active when bound to cyclin)
Can only phosphorylate specific consensus motifs on targets
Levels maintained the same throughout the cycle

Maturation promoting factor

First discovered in frogs
Fusion of mitotic cells with a cell in any other cycle stage causes premature chromosome condensation - these cells go into mitosis
MPF consists of Cyclin and Cdk

Cyclin expression

Different types expressed at different points in the cell cycle
Confer substrate specificity for CDK
Downstream targets of Cyclin-CDK move the cell cycle forward
Levels decrease sharply after the end of the phase - sharp line between phases

Internal influences of cell cycle

Growth
DNA replication
DNA damage
- Little damage must be repaired
- Lots of damage causes senescence or apoptosis

External influences of the cell cycle

Food
Space
Communication with organs - tissue damage and developmental stage
- Hormones

Cyclin destruction

Through ubiquitination
Causes abrupt decrease in Cdk function after end of phase

Ubiquitin-Proteasome System

Ubiquitin - small PTM, covalently linked
Polyubiquitation formed on lysines
An entire protein enters the proteasome and amino acids leave
These can then be used to make more proteins
Ubiquitins open the "lid" of the proteasome so it opens and degrades the protein

APC/C

Anaphase Promoting Complex or Cyclosome
In the ubiquitin ligase family (joins ubiquin to something)
Key regulator of metaphase to anaphase transition
Cyclin is the most important target
Coactivators:
- Cdc20 (mitosis to metaphase)
-- polybiquinates M cyclin (bound to Cdk) and causes degradation
- Cdh1 (anaphase to the start of S phase)

Cohesin

Holds chromosomes together
Cohesin cut by seperase - to go from metaphase to anaphase
Seperase held by securin (inhibitory)
APC ubiquitinates scurin and so releases separase when all chromosomes are lined up in middle of cell

CKI (CDK inhibitor proteins)

Block Cdk function by covering the protein surface (including active site) or covalently attaching chemical groups
Covers the active site of the protein
- Temporary non-covalent interaction
- When DNA damage will stop Cdk from acting and holding in the current cell cycle phase
Is broken down by ubiquintation by the SCF complex
Two families in mammals:
- CIP/KIP
- INK4

Cdk activation

Cyclin binding is necessary but not sufficient
PTMs act to fine regulate
Active site of Cdk only fully active when phosphorylated by cyclin activating kinase (CAK)

Wee1 and Cdc25

Wee1 - inhibitory kinase
Phosphorylates a neighouring site and blocks the active site
Cdc25 - activatory phosphatase
Removes the inhibitory phosphate

p21

A Cdk inhibitor
Covers the cyclin CDK
Regulated by p53
- p53 is often gone in cancer cells

p53

TF to activate p21 transcription
In response to DNA damage, p53 is activated by phosphorylation
Pauses the cell cycle to repair the damage

Oncogenes

Have a normal function in the cell, when upregulated causes cancer
e.g. E2f or Cyclin E

Tumour supressor genes

When missing on not being created causes cancer
e.g. Rb

Positive feedback loop (Mitosis)

Ensure a process keeps going
Activating the activator and suppressing the suppressor

Cell cycle checkpoints

To ensure there is suitable cell state and environment before proceeding to the next stage
Each checkpoint requires a different stimulant and acts through a different Cyclin-Cdk couple
G0/G1 = Mitogen stimulation
G1/S = Restriction point
S/G2 = DNA damage
G2/M = Antephase checkpoint
M/G1 = SAC

Mitogen stimulation

Mitogen = a molecule that pushes the cell into mitosis
Without this it will not enter the cell cycle (will remain in G0)
e.g. growth factors

Restriction point

The point at which the cell decides whether to go through with mitosis
Mitogen signal acts through G1 and G1/S Cdks - phosphorylating Rb and releasing the Rb targets
Rb covers the transcription factor target E2F
The uncovering of this causes the transcription of genes needed for cell proliferation

DNA damage mediated arrest

Can occur at any time in the cell cycle
After a cascade of signals and sequential phosphorylations, Cdk-cyclin complexes are inhibited - through recruitment of CKIs

Spindle Assembly Checkpoint (SAC)

Activated upon nuclear envelope breakdown
To prevent premature segregation of sister chromatids and therefore ensure genome stability
Mitotic checkpoint complex (MCC) is the SAC effector molecule, generated at unattached kinetochores

Mitotic checkpoint complex (MCC)

iThe SAC effector molecule, generated at unattached kinetochores
Inhibits APC/C in order to prevent premature anaphase and unequal segregation of DNA
No longer generated with kinetochores are attached, so APC/C can degrade key substrates

Flow cytometry

Label DNA in samples with dye then fix and sort

Immunohistochemistry (IHC)/Immunofluorescence IF)

Antibodies raised against known mitotic markers
Used to observe cell cycle stage

Cytokinesis

Cytoplasmic division
Roughly in half

Sister Chromatids

Identical chromosome pairs
Often exchange material during interphase

Meiosis

Produce 4 haploid cells from one diploid cell
Two cell divisions

Meiosis I

Reduction division stage
2 haploid from 1 diploid
Interphase I - replication of DNA
Prophase I
- Chromatin coils
- Homologous chromosomes pair up and chromatins intertwine
- Formation of chiasmata - attachments between homologous chromosomes
- Chromosomes begin to move to centre, spindle apparatus begins to form
- Nuclear membrane disappears
Metaphase I
- Spindle formation
- Chromosomes align - two centromeres on opposite side of equatorial plane
Anaphase I
- Chiasmata disappear
- Homologous chromosomes are pulled by spindle fibres to opposite parts of the cell (one of each pair of autosomes and one of sex chromosomes)
Telophase I
- Chromosomes reach opposite ends of cell + slightly uncoil
- Nuclear membrane begins to form
- Cytokinesis (males = equal division, females = unequal - one is polar body)

Meiosis II

Equatorial divison
Each haploid cell replicated
Interphase II - V brief, no replication
Prophase II
- Chromosomes thicken and coil
- Nuclear membrane disappears
- Spindle fibers form
Metaphase II
- Spindle fibers pull chromosomes to equatorial plane
Anaphase II
- Centromeres split and carry single chromatid to either side
Telophase II
- Chromosomes begin to uncoil
- Nuclear membranes formed
- Cytokinesis (males = equally, females = unequal - one becomes polar body(

Spermatogenesis

Constantly occurring
Spermatogonia = diploid
Mitosis -> primary spermatocyte (diploid)
Meiosis I -> 2 secondary spermatocytes (haploid - 23ds)
Meiosis II -> 4 spermatids (haploid - 23ss)
Spermatids lose cytoplasm and develop tails

Oogenesis

Mostly before birth
Oogonia = diploid
Mitosis -> primary oocyte (diploid) - during foetal development
Held in prophase I until birth, continues when ovulated
Meiosis I -> 1 secondary oocyte + 1 polar body (haploid - 23ds)
Emerges from follicle and proceeds down fallopian tube w/ polar attached
Meiosis II -> 1 mature ovum + 1 polar body (haploid - 23ss)
- Only happens if fertilised by a sperm
Polar bodies disintegrate